It is now well recognized that several protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases) play crucial roles in TCR-mediated T cell activation. Previous studies supported by this grant addressed the role and regulation of the Syk family PTKs ZAP-70 and Syk, and substrates for these kinases. We propose to continue this work by focusing on a new ZAP-70 substrate, the 20-kDa Vaccinia virus VH1-related (VHR) dual-specific phosphatase. We propose: (1) To study VHR tyrosine phosphorylation and establish whether VHR is a direct substrate for ZAP-70. We have found that VHR accumulates in the immune synapse in Ag-specific T cells and is phosphorylated on tyrosine in T lymphocytes, as well as in T cell lines, except those lacking ZAP-70 or ZAP-70 activation by Lck. We wish to continue these experiments by verifying that VHR indeed is phosphorylated directly by ZAP-70. We also plan to examine the site, stoichiometry, and time-course of this phosphorylation, and which stimuli and T cell populations that employ it. (2) To determine the role of VHR phosphorylation for ZAP-70 signaling. It is not yet clear what tyrosine phosphorylation does to VHR, but all results point to a functionally activating effect. We will examine the effects of VHR phosphorylation on MAP kinases and other downstream pathways and determine if it affects the subcellular location, nuclear import, interaction with other proteins, or catalytic activity of VHR. (3) To examine the association of VHR with POZ-domain protein that interacts with VHR (POZtiv). We recently identified this novel VHR-binding protein using the yeast two-hybrid system. We hypothesize that it acts as a scaffold protein for VHR, but releases phospho-VHR. Alternatively, it may be required for some other aspect of VHR biology. These issues will be examined. (4) To study the physiological role of VHR in T cell activation. Finally, we plan to generate T cells and mice that lack VHR to determine how important VHR is. To achieve this goal we will utilize three different approaches, each with its own limitations, namely RNA interference (RNAi), antisense, and mouse knock-outs. These experiments will ask how the absence (or severe reduction) of VHR affects TCR-induced activation of MAP kinases, T cell proliferation, effector functions and immune responses in whole animals.